Topoisomerase activity of the hyperthermophilic replication initiator protein Rep75

Abstract

The plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi replicates via the rolling circle mechanism. pGT5 encodes the replication initiator protein Rep75 that exhibits a nicking-closing (NC) activity in vitro on single-stranded oligonucleotides containing the pGT5 double-stranded origin (dso) sequence. Some mesophilic Rep proteins present site-specific DNA topo-isomerase-like activity on a negatively supercoiled plasmid harbouring the dso. We report here that Rep75 also exhibits topoisomerase activity on a negatively supercoiled DNA substrate. This DNA topoisomerase-like activity is dependent on the amino acids involved in NC activity of Rep75. However, in contrast with mesophilic Rep proteins, Rep75 topoisomerase activity is not dso dependent. Moreover, although pGT5 is known to be relaxed in vivo, Rep75 was not able to act on a relaxed plasmid in vitro, whether or not it contained the dso.

Figure 1

pKSG1 and pKSG2 plasmidic maps. The two plasmid constructions are presented in Materials and Methods. The pBSKS+ vector is indicated by grey lines and the pGT5 insert by black lines. The rep75 gene is indicated by an arrow. Whites boxes indicate the two pGT5 origins, sso and dso.

Figure 2

In vitro Rep75 topoisomerase-like activity on the pKSG1 plasmid. The different DNA plasmidic forms are indicated: OC, open circular plasmid; SC, negatively supercoiled plasmid; R, relaxed plasmid at 75°C; L, linear form. c, control samples without Rep75 protein. (A) Topoisomerase reaction kinetics. pKSG1 (100 ng) was incubated with Rep75 at 75°C for the indicated time. (B) Concentration dependence of the topoisomerase reaction on negatively supercoiled plasmid. pKSG1 (100 ng) was incubated with Rep75 at the ratio indicated for 10 min at 75°C. (C) Temperature dependence of Rep75 topoisomerase activity. pKSG1 (100 ng) was incubated with Rep75 for 10 min at the indicated temperature.

Figure 3

Rep75 topoisomerase activity on different substrates. An aliquot of 100 ng of plasmid pKSG1 or pKSG2, negatively supercoiled (S) or relaxed (R), were incubated with (+) or without (–) 100 ng of Rep75 (protein:DNA ratio of 50:1). The incubations were performed for 15 min at 75°C and the reaction products were loaded on an agarose gel in the absence (A) or in the presence (B) of 10 ng/ml of EtBr. Different DNA plasmidic forms are indicated: OC, open circular plasmid; SC, negatively supercoiled plasmid; R, relaxed plasmid at 75°C.

Figure 4

Topoisomerase activities of Rep75 mutated proteins. (A) Sequence of the Rep75 active site. The residues conserved in the active site of different RC replication initiator proteins are indicated in bold (see Fig. 5). The amino acids targeted for substitution are indicated by an asterisk. (B) Activities of Rep75 mutated proteins. The names of the proteins correspond to the mutations they harbour. For the assays performed on supercoiled plasmids, 100 ng of pKSG1 was incubated with 100 ng of the indicated protein (protein:DNA molar ratio of 50:1) for 10 min at 75°C. The oligonucleotide assays have been performed previously (8), and the results are summarised for comparison with topoisomerase activities. The appearance (+) or not (–) of nicking and religation products are indicated. +/– indicates a weak activity. NTT corresponds to the nucleotidyl terminal transferase activity. (C) Activities of three mutated proteins. The experiment was performed as previously described and the BET stained gel is presented. –, control samples without Rep75 protein. W, incubation with Rep75; R, incubation with Rep75-R451L; Y, incubation with Rep75-Y448F; YR, incubation with Rep75-R451L/Y448F.

Figure 4

Topoisomerase activities of Rep75 mutated proteins. (A) Sequence of the Rep75 active site. The residues conserved in the active site of different RC replication initiator proteins are indicated in bold (see Fig. 5). The amino acids targeted for substitution are indicated by an asterisk. (B) Activities of Rep75 mutated proteins. The names of the proteins correspond to the mutations they harbour. For the assays performed on supercoiled plasmids, 100 ng of pKSG1 was incubated with 100 ng of the indicated protein (protein:DNA molar ratio of 50:1) for 10 min at 75°C. The oligonucleotide assays have been performed previously (8), and the results are summarised for comparison with topoisomerase activities. The appearance (+) or not (–) of nicking and religation products are indicated. +/– indicates a weak activity. NTT corresponds to the nucleotidyl terminal transferase activity. (C) Activities of three mutated proteins. The experiment was performed as previously described and the BET stained gel is presented. –, control samples without Rep75 protein. W, incubation with Rep75; R, incubation with Rep75-R451L; Y, incubation with Rep75-Y448F; YR, incubation with Rep75-R451L/Y448F.

Figure 5

Alignment of Rep75 putative active site with those of different RC replication initiator proteins. The residues conserved between all the proteins are indicated in bold characters. The names indicated refer to the plasmid or the phage name: BGMV, Bean Golden Mosaic Geminivirus; MVM, Minute Virus of Mouse.